Addition of inhibitors of chromaffin vesicle catecholamine uptake, reserpine or tetrabenazine, to cultures of bovine adrenal chromaffin cells led to a depletion of cellular catecholamine stores and to an increased opioid peptide content. Addition of monoamine oxidase inhibitors, catecholamines or catecholamine receptor agonists to the cultures produced similar increases in the opioid peptide content without altering catecholamine stores, suggesting that an extravesicular catecholamine pool influences chromaffin cell opioid peptide content. This elevated peptide content resulted from increased synthesis by the cells and led to enhanced stimulus-evoked secretion of opioid peptides. Dissociated cells obtained from a human pheochromocytoma tumor also increased their opioid peptide content following treatment with reserpine or a secretagogue. The results obtained with cultured chromaffin and pheochromocytoma cells suggest that a mechanism for replenishing and increasing adrenal medullary opioid peptide stores and secretory capacity may function in vivo. The observed regulation of opioid peptide stores may also be important in the clinical pathology of opioid peptide containing tumors.